SMARCA4-Deficient Thoracic Sarcoma

Modern Pathology (2017) 30, 1422–1432

SMARCA4-deficient thoracic sarcoma: a distinctive clinicopathological entity with undifferentiated rhabdoid morphology and aggressive behavior Jennifer L Sauter1,4, Rondell P Graham1, Brandon T Larsen2, Sarah M Jenkins3, Anja C Roden1 and Jennifer M Boland1

1Division of Anatomic Pathology, Mayo Clinic, Rochester, MN, USA; 2Division of Anatomic Pathology, Mayo Clinic, Scottsdale, AZ, USA and 3Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA

A distinct subset of thoracic sarcomas with undifferentiated rhabdoid morphology and SMARCA4 inactivation has recently been described, and potential targeted therapy for SMARC-deficient tumors is emerging. We sought to validate the clinicopathological features of SMARCA4-deficient thoracic sarcomas. Clinicopathological information was gathered for 40 undifferentiated thoracic tumors with rhabdoid morphology (mediastinum (n = 18), lung (n = 14), pleura (n = 8)). Thymic carcinomas (n = 11) were used as a comparison group. Immunohistochemistry included BRG1 (SMARCA4), BRM (SMARCA2), INI-1 (SMARCB1), pan-cytokeratin, desmin, NUT, S-100 protein, TTF1, CD34, and SOX2. BRG1 loss was present in 12 of 40 rhabdoid thoracic tumors (30%): 7 of 18 in mediastinum (39%), 2 of 8 in pleura (25%), and 3 of 14 in lung (21%). All BRG1-deficient tumors tested for BRM (n = 8) showed concomitant loss. All thymic carcinomas showed retained BRG1 and INI-1. Morphologically, tumors with BRG1 loss showed sheets of monotonous ovoid cells with indistinct cell borders, abundant eosinophilic cytoplasm, and prominent nucleoli. Scattered areas with rhabdoid morphology (ie, eccentric nuclei, dense eosinophilic cytoplasm, discohesion) were present in all the cases. SMARCA4/BRG1- deficient sarcomas showed rare cells positive for cytokeratin in 10 cases (83%). One showed rare TTF1-positive cells. All were negative for desmin, NUT, and S-100 protein. CD34 was positive in three of five (60%) BRG1- deficient tumors tested. SOX2 was positive in all four BRG1-deficient tumors tested, and negative in all seven tested cases with retained BRG1. SMARCA4/BRG1-deficient sarcomas occurred at median age of 59 years (range 44–76) with male predominance (9:3) and had worse 2-year survival compared with BRG1-retained tumors (12.5% vs 64.4%, P = 0.02). SMARCA4-deficient thoracic sarcomas can be identified based on their distinctive high-grade rhabdoid morphology, and the diagnosis can be confirmed by immunohistochemistry. Identification of these tumors is clinically relevant due to their aggressive behavior, poor prognosis, and potential targeted therapy. Modern Pathology (2017) 30, 1422–1432; doi:10.1038/modpathol.2017.61; published online 23 June 2017

The SMARCA4 gene is located on chromosome 19p complex is formed by multiple proteins, of which and encodes the BRG1 protein, one of the two INI-1 (encoded by SMARCB1 gene) is the best mutually exclusive catalytic subunits of the switch/ characterized. The complex acts as a tumor suppres- sucrose-nonfermenting (SWI/SNF) chromatin-remo- sor by regulating transcription and promoting cell 1–8 deling complex. The other catalytic subunit is BRM, differentiation. Inactivation of SMARCA4 has encoded by the SMARCA2 gene. The SWI/SNF been reported in several aggressive tumors with high-grade undifferentiated rhabdoid morphology, including small cell carcinoma of the ovary, hyper- Correspondence: Dr JM Boland, MD, Division of Anatomic calcemic type (SCCOHT), and a subset of atypical Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN teratoid–rhabdoid tumors of the central nervous 55905, USA. system.9–15 Carcinomas from various sites have E-mail: [email protected] also been shown to have SMARCA4 inactivation 4Current address: Department of Pathology, Memorial Sloan including those arising in the endometrium, gastro- Kettering Cancer Center, New York, NY, USA. 16–26 Received 31 March 2017; revised 27 April 2017; accepted 27 April intestinal tract, and lung. Morphologically, 2017; published online 23 June 2017 carcinomas with SMARCA4 inactivation include

www.modernpathology.org SMARCA4/BRG1 loss in thoracic sarcomas JL Sauter et al 1423 both differentiated carcinomas (adenocarcinomas Materials and methods and squamous cell carcinomas), and undifferentiated carcinomas with variably rhabdoid morphology. Case Selection SCCOHT is the prototypic rhabdoid-appearing Following institutional review board approval, two tumor with SMARCA4 inactivation, and has a histo- thoracic pathologists (JLS and JMB) retrospectively logic phenotype morphologically similar to the better reviewed hematoxylin and eosin-stained slides from characterized SMARCB1/INI-1-deficient tumors. This mediastinum, pleura and lung tumors from institu- expandinggrouphasbeenreferredtoas‘SMARCB1- tional surgical and consultation archives, identified deficient neoplasia’,27 and includes atypical teratoid– using search terms that included ‘rhabdoid’, ‘undif- rhabdoid tumors,28 rhabdoid meningiomas,29 malig- ferentiated’,or‘poorly differentiated malignant neo- nant rhabdoid tumors (MRT) of the kidney and soft plasm’. Undifferentiated rhabdoid tumors encou- tissue,30–32 rhabdoid carcinomas of the gastrointest- ntered in our consultation practice were also inal tract,33 pancreatic undifferentiated rhabdoid prospectively included. Possible or known metastatic carcinomas,34 and epithelioid sarcomas.35,36 These tumors from extra-thoracic primary sites were tumors share overlapping morphologic features excluded, as were cases with an alternative definitive including rhabdoid histology, loss of INI-1 by immu- diagnosis (eg, NUT carcinoma, differentiated carci- nohistochemistry, focal cytokeratin/EMA immunoex- noma, rhabdomyosarcoma, melanoma, etc) estab- pression, and occasional positive immunostaining for lished by clinical or study immunohistochemical work-up. Study cases (n = 40) with undifferentiated desmin and CEA.27 Inactivation of alternative SWI/ rhabdoid morphology were selected. A group of SNF complex members, such as SMARCA4 and surgically resected thymic carcinomas (n = 11) con- SMARCA2, may explain the molecular pathway firmed by morphology and immunohistochemistry underlying tumors histologically similar to were included as a comparison group given their SMARCB1/INI-1-deficient tumors, but with retained mediastinal location, including nine squamous cell INI-1 immunoexpression. 23 carcinomas, one adenocarcinoma, and one undiffer- In a recent genetic study, Le Loarer et al identified entiated carcinoma. a group of undifferentiated thoracic malignancies Clinical information was obtained from the elec- with SMARCA4 inactivation presenting as compres- tronic medical record and from referring physicians, sive tumors often involving the mediastinum, with or if available. Selected clinical parameters were without lung involvement, occurring in young recorded, including demographics, clinical presenta- patients and displaying aggressive behavior. They tion, location and size of tumor at presentation, type found these tumors to be genetically distinct from of surgical procedure, treatment, and outcome. lung carcinomas with SMARCA4 deficiency, and to display a closer molecular relationship to SCCOHT and MRT. In contrast to lung carcinomas with isolated Immunohistochemistry SMARCA4 inactivation, these SMARCA4-deficient thoracic malignancies also showed co-deficiency of Immunohistochemistry for BRG1 was performed on SMARCA2. Because of these clinicopathological and whole paraffin-embedded tissue sections from the 40 molecular differences, Le Loarer and colleagues rhabdoid thoracic tumors and 11 thymic carcinomas. proposed the term ‘SMARCA4-deficient thoracic Following EDTA antigen retrieval, monoclonal BRG1 sarcomas’ for this distinctive entity.23 A subsequent antibody (clone EPNCIR111A, Abcam, Cambridge, study further confirmed the unique molecular and MA, USA) was used at 1:200 dilution (15 min clinicopathological features of SMARCA4-deficient incubation) with polymer refine detection (Leica thoracic sarcomas in a Japanese cohort.22 DS9800). Negative and positive controls were per- Currently, there is a clinical trial available for formed with each run. Immunostaining was also patients with SMARC-deficient tumors with an performed on all undifferentiated rhabdoid tumors, inhibitor to the histone-lysine N-methyltransferase using antibodies directed against INI-1 (clone 25/ Enhancer of Zeste Homolog 2 or EZH2,37 and BAF47, BD Transduction Laboratories, San Jose, CA, USA), desmin (clone DE-R-11, Novocastra, New alternative targeted therapies are under development – Castle, UK), NUT (clone C52B1, Cell Signaling for tumors deficient in SMARCA4 expression.38 43 Technology, Danvers, MA, USA), S-100 protein Given the potential therapeutic implications, it is (Dako, Glostrup, Denmark), and TTF1 (clone important to identify SMARCA4-deficient thoracic SPT24, Novocastra). Pan-keratin clones included sarcomas, including diagnosis from small biopsies. OSCAR keratin (Biolegend, San Diego, CA, USA) In this study, our main objectives were to determine and/or keratin AE1/AE3 (Dako). Immunostains for the frequency of SMARCA4 inactivation in CD34 (Novocastra), BRM, and SOX2 (clone EPR3131, thoracic sarcomas with undifferentiated rhabdoid Abcam) were performed on subsets of the rhabdoid morphology, and to further validate the clinico- tumors with tissue available for additional immuno- pathological features of these tumors in a Western histochemistry. Following citrate antigen retrieval, a population. polyclonal BRM antibody (Sigma-Aldrich, St. Louis,

Modern Pathology (2017) 30, 1422–1432 SMARCA4/BRG1 loss in thoracic sarcomas 1424 JL Sauter et al

Table 1 Clinicopathologic features of BRG1-deficient/retained thoracic tumors and thymic carcinomas

BRG1-deficient tumors BRG1-retained tumors Thymic carcinomas Features (n = 12) (n = 28) (n = 11)

Median age (yrs), (range) 59 (44–76) 54.5 (19–84) 51 (32–79) Male 9 (75%) 22 (79%) 6 (55%)

Site Mediastinum 7 (58%) 11 (39%) 11 (100%) Lung 3 (25%) 11 (39%) — Pleura 2 (17%) 6 (22%) —

Metastatic disease 7/9 (78%) 10/22 (45%) 6/11 (55%) Unresectable tumor 6/6 (100%) 6/16 (38%) 6/11 (55%)

Treatment, n 61611 Surgery only 4 (67%) 3 (19%) 0 Surgery+chemo 2/6 (33%) 3 (19%) 0 Surgery+XRT 1 (6%) 3 (27.3%) Surgery+chemo+XRT 3 (19%) 2 (18.2%) Chemo only 2 (12%) 1 (9%) XRT only 1 (6%) 2 (18.2%) Chemo and XRT 3 (19%) 3 (27.3%)

Available follow-up, n 81811 Median (range) follow-up (mos) 4.0 (0.7–108.5) 17.4 (0.6–183.6) 34.3 (2.3–162.8) Outcome, n 8 DOD 9 DOD 8 DOD

Abbreviations: Chemo, chemotherapy; DOD, dead of disease; mos, months; XRT, radiation therapy; yrs, years.

MO, USA) was used at 1:400 dilution on Leica Bond Thirteen patients had a history of smoking, 1 patient III stainer. was a never smoker, and the smoking status of the Loss of BRG1, INI-1, and BRM immunoexpression remaining patients was not known. was defined as complete unequivocal loss of expression in tumor nuclei, in the presence of positive internal control (eg, background inflammatory cells, Immunohistochemistry for SWI/SNF Complex Proteins stromal fibroblasts, endothelial cells, or benign Analysis of SMARCA4 expression based on BRG1 epithelial cells). Heterogeneous patterns of retained immunohistochemistry was performed in all the BRG1, INI-1, or BRM immunoexpression with cell- cases. Of the 40 undifferentiated/rhabdoid thoracic to-cell variability in expression were scored as tumors, loss of BRG1 immunoexpression was present ‘retained’. in 12 (30%): 7 of 18 in the mediastinum (39%), 2 of 8 in the pleura (25%), and 3 of 14 in the lung (21%; Table 1). Eight of the tumors with BRG1 loss had Statistical Analysis sufficient tissue available to evaluate for BRM The median survival and 2-year overall survival (SMARCA2) co-deficiency, which was present in all were estimated with the Kaplan–Meier method. the cases tested (Table 2; Figures 1a and b). Isolated Survival was compared with likelihood ratio tests. SMARCA2 inactivation was also seen in one additional mediastinal tumor with undifferentiated rhabdoid morphology that showed retained BRG1 and INI-1 immunoexpression (Table 2). INI-1 staining Results was performed in all the cases, and loss was seen in Clinicopathological Characteristics of Study one pleural tumor with retained BRG1 immunoex- Population pression, while all the others demonstrated retained INI-1 (Table 2; Figure 1c). All thymic carcinomas Forty primary thoracic tumors with undifferentiated showed retained BRG1 and INI-1. Several cases rhabdoid morphology were included: 18 from the (n = 7; three mediastinal tumors, two pleural tumors, mediastinum, 14 from the lung, and 8 from the one lung tumor and one thymic carcinoma) showed pleura (Table 1). Specimens included 21 surgical heterogeneous patterns of nuclear positivity for excisions/biopsies and 19 core or transbronchial BRG1, characterized by cell-to-cell variation in biopsies. Eleven surgically resected thymic carcino- BRG1 expression within the tumor. Since the mas were also included as a comparison group. significance of ‘mosaic’ expression is unknown, the Altogether, the study cohort included 37 men and 14 immunoexpression in these cases was interpreted as women, with a median age of 56 years (range 19–84). retained.

Modern Pathology (2017) 30, 1422–1432 Table 2 Clinical data for patients with thoracic tumors showing loss of BRG1, BRM, or INI-1 immunoexpression

Time to Age Presenting death Patient (yrs) Sex Site (size) BRG1 BRM INI-1 symptoms Imaging features Biopsy type Treatment Outcome (mos)

1 69 F Mediastinum L L R Pain from iliac bone Superior anterior mediastinum mass, Surgical Unresectable, chemo DOD 4 (NA) mets mediastinal LAD, pelvis and iliac +XRT bone mets 2 66 M Mediastinum L R R Dyspnea Anterior mediastinal/hilar mass, Needle Unresectable, chemo; DOD 4 (9.6 cm) echogenic area in pericardium progression on 2 regimens 3 44 M Mediastinum L R R NA Mediastinal mass, mediastinal LAD, Surgical Unresectable, chemo DOD 1 (NA) axilla and lung mets 4 56 M Mediastinum L L R Wt loss, fatigue, Mediastinal/hilar mass Needle Unresectable, chemo; no DOD 4 (10 cm) reflux response on 2 regimens 5 50 M Mediastinum L L R Dyspnea, chest pain Left mediastinal mass encasing major Surgical Unresectable, chemo DOD 2 (16 cm) vessels, severe midline shift, left jugular vein thrombosis 6 48 M Pleura (NA) L L R Pleural effusion NA Surgical NA DOD 108 7 57 M Mediastinum L L R Dyspnea Mediastinal mass Surgical Unresectable, chemo; no DOD 4 (NA) response, subsequent brain and axillary mets 8 61 M Lung (1.3 cm) L NA R NA PET avid (SUV max 3.44) RLL lung Surgical NA NA mass, mediastinal LAD, left adrenal mets 9 65 M Pleura (NA) L L R NA NA Surgical NA NA

10 48 F Mediastinum L NA R NA PET avid (SUV max 12) anterior Needle NA NA Sauter JL SMARCA4 (10 cm) mediastinal mass encasing and narrowing trachea and R main stem

bronchus, supraclavicular LAD al et 11 76 F Lung (NA) L L R NA Solitary RUL lung mass Needle NA DOD 4 sarcomas thoracic in loss /BRG1 12 67 M Lung (1.4 cm) L L R Seizure 1.4 cm RUL nodule, brain and right Surgical NA NA adrenal mets 13 36 M Mediastinum R L R NA NA Surgical NA NA (NA) 14 60 M Pleura (NA) R NA L Pleural effusion, NA Surgical NA NA LLL lung consolidation

Abbreviations: Chemo, chemotherapy; DOD, dead of disease; dx, diagnosis; F, female; L, lost; LAD, lymphadenopathy; LLL, left lower lobe; M, male; mets, metastases; mos, months; NA, not available; PET, positron emission tomography; R, retained; RLL, right lower lobe; RUL, right upper lobe; SUV max, maximum standard uptake value; wt, weight; XRT; radiation therapy; yrs, years. oenPathology Modern 21)3,1422 30, (2017) – 1425 1432 SMARCA4/BRG1 loss in thoracic sarcomas 1426 JL Sauter et al

Figure 1 SMARCA4-deficient thoracic sarcoma demonstrating loss of BRG1 (SMARCA4) immunostaining in all tumor nuclei, with internal control demonstrated by staining of inflammatory and stromal cell nuclei (a; × 200). BRM (SMARCA2) immunoexpression is also lost, with positive internal control (b; × 200). INI-1 (SMARCB1) immunoexpression is retained (c; × 400). Solid architecture is seen in SMARCA4-deficient thoracic sarcomas (d; × 100). A region with more classic rhabdoid morphology, characterized by discohesive cells with eccentric displacement of nuclei by condensation of eosinophilic cytoplasm (e; × 200 and f; × 400). Ovoid tumor cells with abundant eosinophilic cytoplasm, vesicular chromatin, and prominent nucleoli (g; × 200). An example of positive cytokeratin (OSCAR) immunoexpression in only rare tumor cells (h; × 200).

Clinicopathological and Immunohistochemical for two tumors, both of which were PET avid (SUV Features of SMARCA4-Deficient Thoracic Sarcomas max 3.44 and 12). The lung tumors presented at smaller sizes (1.3 and 1.4 cm in the two patients with SMARCA4/BRG1-deficient thoracic sarcomas available size), but both had metastatic disease at the (n = 12) occurred at median age of 59 years (range, – time of the presentation (adrenal (both patients), 44 76), with male predominance (9:3). The epicenter brain and mediastinal lymph nodes). In total, six of disease was mediastinum in 7 patients, lung in 3, patients with BRG1-deficient tumors had known and pleura in 2. The smoking status was known for evidence of metastases at presentation, and one six patients, five of whom were either current or developed progressive metastases 1 month after former smokers. Presenting symptoms included diagnosis. The sites of metastatic disease included dyspnea, chest pain, pleural effusion, reflux, fatigue, lymph nodes (mediastinal, axillary, and supraclavi- and weight loss. One patient presented with right cular), adrenal, pericardium, lung, bone, and brain. lower extremity pain and weakness due to metas- The morphology of the tumors was similar among tases in the iliac bone, and one presented with all the cases and had a characteristic appearance. A seizures due to brain metastases (Table 2). striking feature observed in all the cases was a Of six patients with detailed presentation data, all relatively monotonous appearance of the tumor cells, presented with unresectable tumors. The tumor size especially when considering their otherwise high- was available in four mediastinal tumors, and ranged grade histological characteristics. Secondary or from 9.6 to 16 cm (Table 2). PET data was available complex architectural structures were not present;

Modern Pathology (2017) 30, 1422–1432 SMARCA4/BRG1 loss in thoracic sarcomas JL Sauter et al 1427

Table 3 Overall survival among patients with follow-up

N Deaths Median survival (mo) 2-Year survival % (95% CI) Hazard ratio (95% CI)

BRG1-deficient 8 8 4.0 12.5% (0.0%, 35.4%) 2.86 (1.02, 8.04) BRG1-retained 18 9 39.9a 64.4% (38.7%, 90.1%) 0.87 (0.33, 2.33) Thymic carcinoma 11 8 36.3b 63.6% (35.2%, 92.1%) Reference aSurvival of BRG1-deficient vs BRG1-retained: P = 0.02. bSurvival of BRG1-deficient versus thymic carcinoma: P = 0.06. rather, the tumor cells formed solid sheets or vaguely the thymic carcinoma group (median 36.3 months; anastomosing islands or nodules of tumor cells range 2–54; Tables 1–3). Patients with SMARCA4/ (Figure 1d and g). The tumor cells in many cases BRG1-deficient thoracic sarcomas had worse 2-year displayed a syncytial-like growth pattern, with survival compared with those with BRG1-retained indistinct cell borders. They generally had vesicular tumors and those with thymic carcinomas (12.5% vs chromatin with prominent moderately sized 64.4% and 63.3%, respectively; Table 3). The overall nucleoli. Careful examination typically revealed survival of patients with SMARCA4/BRG1-retained variably represented areas of more classic rhabdoid tumors and thymic carcinomas was similar, but the morphology, including discohesive ovoid tumor overall survival of patients with SMARCA4/BRG1- cells with abundant eosinophilic cytoplasm, deficient thoracic sarcomas trended toward worse eccentric nuclei, and vague perinuclear hyaline survival in comparison among all three groups inclusions (Figures 1e–f). Brisk mitotic activity (Figure 2a). In pairwise comparisons of survival (Figure 1f) and necrosis were universal. between the study subgroups, the overall survival of BRG1-deficient cases showed rare tumor cells with patients with tumors with SMARCA4/BRG1 loss was cytokeratin expression in 10 cases (83%; Figure 1h). significantly worse than those with retained However, diffuse positivity for cytokeratin was not SMARCA4/BRG1 immunoexpression (P = 0.02) and seen in any of the BRG1-deficient cases. One BRG1- approached significance when compared with the deficient case showed weak TTF1 immunoexpres- overall survival of the patients in the thymic sion, and cytokeratin was negative in this case. All carcinoma group (P = 0.06; Figures 2b and c). There BRG1-deficient cases were negative for desmin, NUT, was no difference in survival between patients with and S-100 protein. CD34 immunostaining was positive thymic carcinoma and those with retained in three of five BRG1-deficient tumors tested (60%). SMARCA4/BRG1 immunoexpression (P = 0.84). SOX2 immunohistochemistry was performed in four cases with BRG1 loss, all of which showed strong and diffuse SOX2 expression; SOX2 was negative in seven Discussion tested cases with retained BRG1 expression. In this study, we have demonstrated that SMARCA4/ BRG1 deficiency characterizes a subset of undiffer- Clinical Outcome entiated thoracic tumors with rhabdoid morphology and aggressive behavior. Sarcomas with rhabdoid In total, follow-up information was available for 37 morphology in the chest appear more likely to show patients (median, 12 months; range, 0.5–184), SMARCA4 inactivation, often with co-inactivation of including 8 with BRG1 loss, 18 with retained SMARCA2, rather than inactivation of SMARCB1, BRG1, and 11 thymic carcinomas (Table 3). Known and have a propensity for the mediastinum, although metastases occurred in 7 patients with SMARCA4- they also occur in the pleuropulmonary parench- deficient thoracic sarcomas, 10 with BRG1-retained yma. This work expands on the prior studies in tumors, and 6 thymic carcinomas (Tables 1 and 2). French and Japanese cohorts of SMARCA4-deficient All patients (n = 6) with SMARCA4/BRG1-deficient thoracic sarcomas22,23 by further contributing to the thoracic sarcomas and available data had unresect- clinicopathological characterization of these tumors. able tumors, 4 of whom were treated with che- Similar to the findings of Yoshida et al,22 our cohort motherapy only, and 2 received both chemotherapy showed a male predominance with a wide age range and radiation therapy (Tables 1 and 2). Six of 16 (second to eighth and fourth to seventh decades in patients with BRG1-retained tumors and 6 of 11 the Japanese and our cohorts, respectively). How- patients with thymic carcinoma presented with ever, in both the Japanese and French cohorts, these unresectable tumors, and were treated with a variety sarcomas occurred in younger patients (medians of of modalities (Table 1). 39 and 42 years, respectively) compared with our All patients with SMARCA4/BRG1-deficient cohort (median 59 years). Our findings indicate that sarcomas and available follow-up (n = 8) were dead it is important to consider SMARCA4-deficient of disease at a median of 4 months (range, 1–108), thoracic sarcoma in the differential diagnosis of compared with 9 of 18 in the BRG-retained group tumors showing suggestive morphologic features in (median 39.9 months; range 0.5–96), and 8 of 11 in patients of all ages, as in our experience, this entity is

Modern Pathology (2017) 30, 1422–1432 SMARCA4/BRG1 loss in thoracic sarcomas 1428 JL Sauter et al

Figure 2 Overall survival of patients with BRG1-deficient tumors trends toward worse survival when comparing overall survival of patients with thymic carcinoma, BRG1-retained tumors, and BRG1-deficient tumors (a). In pairwise comparisons of survival between study subgroups, the overall survival of patients with BRG1-deficient tumors was significantly worse than those with retained BRG1 (P = 0.02) (b), and approaches significance when compared with the thymic carcinoma group (P = 0.06; c).

not limited to young patients. In all the three cohorts, lung carcinomas. Le Loarer et al23 demonstrated that the tumors occurred predominantly in the mediasti- SMARCA4-deficient thoracic sarcomas are geneti- num and showed a nonspecific immunophenotype cally distinct from lung carcinomas and are instead with many cases showing very focal, but not diffuse, more closely related to MRTs and SCCOHTs. In their cytokeratin expression. The median survival in both study, the SMARCA4-deficient thoracic sarcomas the French and Japanese groups was 7 months and also showed co-deficiency in SMARCA2, while lung was similarly poor in our cohort at only 4 months. carcinomas showed isolated SMARCA4 inactivation. The findings of our study support and further Co-deficiency of these genes has also been identified validate the behavior of SMARCA4-deficient thoracic in SCCOHT.44 Herpel et al24 found isolated sarcomas as very aggressive, conferring an extremely SMARCA4 loss in 10 of 316 (3.2%) differentiated poor prognosis. lung carcinomas, but co-deficiency of SMARCA4 and An interesting question requiring further study is SMARCA2 in only 0.1% (2 squamous cell the relationship, or lack thereof, between SMARCA4- carcinomas and 2 adenocarcinomas). All tested deficient thoracic sarcomas and SMARCA4-deficient BRG1-deficient thoracic tumors in our study showed

Modern Pathology (2017) 30, 1422–1432 SMARCA4/BRG1 loss in thoracic sarcomas JL Sauter et al 1429 co-inactivation of SMARCA4 and SMARCA2 at the also very similar to the pattern expected for lung protein level. This concomitant loss of both carcinoma (mediastinal lymph nodes, adrenal, bone, SMARCA4 and SMARCA2 expression, in addition and brain), although that is not a specific feature. to the undifferentiated rhabdoid morphology and One might argue for classification of SMARCA4- immunophenotype seen in these tumors, support deficient thoracic tumors as carcinomas based on the that the tumors described herein fall into the focal keratin expression that is often observed; category of SMARCA4-deficient thoracic sarcomas however, focal keratin expression is also commonly rather than lung carcinomas with SMARCA4 inacti- seen in other tumors with SMARCA2 or SMARCB1 vation. SOX2 immunohistochemistry has been pro- loss that are generally accepted as sarcomas. In posed as a marker for distinguishing SMARCA4- addition, as mentioned above, genetic data indicate deficient thoracic sarcomas from carcinomas with that these tumors are distinct from lung carcinomas SMARCA4 inactivation.23 In our study, we per- with SMARCA4 inactivation.23 The propensity for formed SOX2 staining in a subset of cases with the mediastinum, overrepresentation of younger BRG1 loss and retained BRG1 expression. SOX2 was patients, and distinctive monotonous morphology diffusely positive in all the tested cases with BRG1 (as opposed to the usual pleomorphism of carci- loss, and negative in the tested cases with retained noma) also support the concept of these tumors as de BRG1 expression, although our number of tested novo and distinct malignancies. Therefore, the cases was small. However, its use as a diagnostic nature of the relationship of these tumors to lung marker in this setting remains to be established, as it carcinoma and/or smoking remains to be deter- is not specific and may be expressed in several mined. It is also possible that the early studies of carcinomas that could enter the differential diagno- this entity have comprised a mix of ‘true’ SMARCA4- sis, including a high percentage of squamous cell deficient sarcomas as well as poorly differentiated carcinomas and high-grade neuroendocrine carcino- carcinomas with SMARCA4 inactivation, as pathol- mas, as well as rare lung adenocarcinomas.45–48 ogists are refining the ability to identify and properly Yoshida et al also observed SOX2 immunostaining classify these rare tumors. in 4 of 12 SMARCA4/BRG1-deficient lung carcino- In addition to prognostic implications, identifica- mas, although only one was diffuse.22 A recent study tion of SMARCA4-deficient thoracic sarcomas is demonstrated that Claudin-4 immunohistochemistry becoming important for clinical management as may be helpful in distinguishing SWI/SNF complex- targeted therapy begins to emerge. Currently, tar- deficient undifferentiated tumors/sarcomas from the geted EZH2 inhibitors are being developed for the various carcinomas that show loss of SMARCA4 treatment of tumors with abnormalities in the SWI/ expression.49 The clinical significance of this dis- SNF complex proteins. EZH2 is a histone methyl- tinction in the thorax remains to be determined, as transferase that acts as the catalytic subunit of the lung carcinomas with SMARCA4 loss also have a polycomb repressor complex (PRC), a transcriptional poor prognosis,50–52 and direct comparative studies regulator involved in cancer metastasis and cell have not been performed. However, as discussed proliferation. The SWI/SNF complex regulates the below, emerging evidence suggests that tumors with PRC, and if this regulation is disturbed by mutations SMARCA4 and SMARCA2 co-deficiency may in the proteins of the SWI/SNF complex, uninhibited respond more favorably to targeted therapy than PRC activity leads to tumor progression and those with isolated SMARCA4 loss. metastasis. Targeted EZH2 inhibitors disable the Currently, it is not entirely clear whether PRC complex and stop uninhibited cell SMARCA4-deficient thoracic sarcomas represent proliferation.59,60 Currently, there is a clinical trial truly unique de novo neoplasms, or possibly a form with an EZH2 inhibitor available for patients with of ‘dedifferentiation’ from a prior lung carcinoma SMARC-deficient tumors,37 including SMARCA4- with or without SMARCA4 inactivation. This is not a deficient thoracic sarcomas demonstrating rhabdoid novel concept, as INI-1/SMARCB1 loss has been morphology. In addition, emerging data suggest that rarely noted to occur as a secondary phenomenon in co-deficiency of SMARCA4 and SMARCA2 expres- malignancies of various types, imparting a ‘compo- sion confers synthetic lethality to EZH2 inhibitors as site extrarenal rhabdoid tumor’ morphology.53–55 preclinical models of SMARCA2- and SMARCA4- Tumors with SMARC-intact differentiated areas deficient SCCOHT, MRT and lung adenocarcinomas juxtaposed with dedifferentiated components defi- have shown selective killing with EZH2 cient in a SWI/SNF protein have also been reported inhibition.61–63 Although clinical trial data are not in several organs.20,56–58 Thus, the fundamental yet available, knowledge of the SMARCA2 status in a classification of these tumors as sarcoma vs carci- tumor may prove to be important not only for noma is still a matter of debate. Some of the distinguishing these sarcomas from SMARCA4-defi- mutations detected by Yoshida et al in their cohort cient carcinomas but also as a predictive biomarker of SMARCA4-deficient thoracic sarcomas are com- for response to therapy. mon in lung adenocarcinoma (eg, KRAS, TP53), and As SMARCA4-deficient thoracic sarcomas usually there was an increased number of smokers and present at an advanced stage and are often unresect- patients with emphysema in their cohort.22 The able, many will be only sampled via minimally distribution of metastases observed in our study is invasive small biopsies. In these situations, BRG1

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and BRM immunostaining may be critical for under- 10 Ramos P, Karnezis AN, Craig DW, et al. Small cell standing the molecular mechanism of these tumors carcinoma of the ovary, hypercalcemic type, displays frequent inactivating germline and somatic mutations and for selecting the appropriate patients for targeted – therapy, even with scant samples that may contain in SMARCA4. Nat Genet 2014;46:427 429. 11 Jelinic P, Mueller JJ, Olvera N, et al. Recurrent insufficient quantity of tumor for more advanced SMARCA4 mutations in small cell carcinoma of molecular testing. the ovary. Nat Genet 2014;46:424–426. In conclusion, SMARCA4-deficient thoracic 12 Karanian-Philippe M, Velasco V, Longy M, et al. sarcomas have distinctive morphological features SMARCA4 (BRG1) loss of expression is a useful marker that can be identified by pathologists. They are for the diagnosis of ovarian small cell carcinoma of the histologically similar to tumors with INI-1 loss and hypercalcemic type (ovarian rhabdoid tumor): a com- show a high-grade rhabdoid appearance, often with prehensive analysis of 116 rare gynecologic tumors, 9 cytokeratin expression in rare tumor cells. BRG1 soft tissue tumors, and 9 melanomas. Am J Surg Pathol – and/or BRM immunohistochemistry is useful to 2015;39:1197 1205. confirm the diagnosis, and an immunohistochemical 13 Clarke BA, Witkowski L, Ton Nu TN, et al. Loss of SMARCA4 (BRG1) protein expression as determined panel is helpful for the exclusion of morphologic by immunohistochemistry in small-cell carcinoma of mimics. The identification of SMARCA4-deficient the ovary, hypercalcaemic type distinguishes these thoracic sarcomas is important both prognostically tumours from their mimics. Histopathology 2016;69: and therapeutically. These tumors behave aggres- 727–738. sively and have a prognosis worse than other poorly 14 Conlon N, Silva A, Guerra E, et al. Loss of SMARCA4 differentiated thoracic tumors. The development of expression is both sensitive and specific for the diagnosis targeted therapy provides additional clinical rele- of small cell carcinoma of ovary, hypercalcemic type. – vance for the identification of SMARCA4-deficient Am J Surg Pathol 2016;40:395 403. thoracic sarcomas by pathologists. 15 Hasselblatt M, Gesk S, Oyen F, et al. Nonsense mutation and inactivation of SMARCA4 (BRG1) in an Atypical Teratoid/Rhabdoid Tumor Showing Retained SMARCB1 (INI1) Expression. Am J Surg Pathol Disclosure/conflict of interest 2011;35:933–935. 16 Strehl JD, Wachter DL, Fiedler J, et al. Pattern of The authors declare no conflict of interest. SMARCB1 (INI1) and SMARCA4 (BRG1) in poorly differentiated endometrioid adenocarcinoma of the uterus: analysis of a series with emphasis on a novel SMARCA4-deficient dedifferentiated rhabdoid variant. References Ann Diagn Pathol 2015;19:198–202. 17 Hoang LN, Lee Y-S, Karnezis AN, et al. Immunophe- 1 Wang W, Xue Y, Zhou S, et al. Diversity and notypic features of dedifferentiated endometrial carci- specialization of mammalian SWI/SNF complexes. noma - insights from BRG1/INI1-deficient tumours. Genes Dev 1996;1:2117–2130. Histopathology 2016;69:560–569. 2 Wong AK, Shanahan F, Chen Y, et al. BRG1, a 18 Stewart CJR, Crook ML. SWI/SNF complex deficiency component of the SWI-SNF complex, is mutated in and mismatch repair protein expression in undiffer- multiple human tumor cell lines. Cancer Res 2000;60: entiated and dedifferentiated endometrial carcinoma. 6171–6177. Pathology 2015;47:439–445. 3 Shain AH, Pollack JR. The spectrum of SWI/SNF 19 Karnezis AN, Hoang LN, Coatham M, et al. Loss of mutations, ubiquitous in human cancers. PLoS ONE switch/sucrose non-fermenting complex protein expres- 2013;8:e55119. sion is associated with dedifferentiation in endometrial 4 Wilson BG, Roberts CW. SWI/SNF nucleosome remo- carcinomas. Mod Pathol 2016;29:302–314. dellers and cancer. Nat Rev Cancer 2011;11:481–492. 20 Donner LR, Wainwright LM, Zhang F, et al. Mutation of 5 Kadoch C, Crabtree GR. Mammalian SWI/SNF chroma- the INI1 gene in composite rhabdoid tumor of the tin remodeling complexes and cancer: mechanistic endometrium. Hum Pathol 2007;38:935–939. insights gained from human genomics. Sci Adv 2015;1: 21 Agaimy A, Daum O, Märkl B, et al. SWI/SNF complex- e1500447. deficient undifferentiated/rhabdoid carcinomas of the 6 Peterson CL, Dingwall A, Scott MP. Five SWI/SNF gene gastrointestinal tract: a series of 13 cases highlighting products are components of a large multisubunit mutually exclusive loss of SMARCA4 and SMARCA2 complex required for transcriptional enhancement. and frequent co-inactivation of SMARCB1 and Proc Natl Acad Sci USA 1994;91:2905–2908. SMARCA2. Am J Surg Pathol 2016;40:544–553. 7 Kadoch C, Hargreaves DC, Hodges C, et al. Proteomic 22 Yoshida A, Kobayashi E, Kubo T, et al. Clinicopatho- and bioinformatic analysis of mammalian SWI/SNF logical and molecular characterization of SMARCA4- complexes identifies extensive roles in human malig- deficient thoracic sarcomas with comparison to nancy. Nat Genet 2013;45:592–601. potentially related entities. Mod Pathol 2017;30: 8 Oike T, Ogiwara H, Nakano T, et al. Inactivating 797–809. mutations in SWI/SNF chromatin remodeling genes in 23 Le Loarer F, Watson S, Pierron G, et al. SMARCA4 human cancer. Jpn J Clin Oncol 2013;43:849–855. inactivation defines a group of undifferentiated thor- 9 Witkowski L, Carrot-Zhang J, Albrecht S, et al. Germ- acic malignancies transcriptionally related to BAF- line and somatic SMARCA4 mutations characterize deficient sarcomas. Nat Genet 2015;47:1200–1205. small cell carcinoma of the ovary, hypercalcemic type. 24 Herpel E, Rieker RJ, Dienemann H, et al. SMARCA4 Nat Genet 2014;46:438–443. and SMARCA2 deficiency in non-small cell lung

Modern Pathology (2017) 30, 1422–1432 SMARCA4/BRG1 loss in thoracic sarcomas JL Sauter et al 1431

cancer: immunohistochemical survey of 316 consecu- 39 Glaros S, Cirrincione GM, Muchardt C, et al. The tive specimens. Ann Diagn Pathol 2017;26:47–51. reversible epigenetic silencing of BRM: implications for 25 Matsubara D, Kishaba Y, Ishikawa S, et al. Lung cancer clinical targeted therapy. Oncogene 2007;26: with loss of BRG1/BRM, shows epithelial mesenchy- 7058–7066. mal transition phenotype and distinct histologic and 40 Kahali B, Gramling SJB, Marquez SB, et al. Identifying genetic features. Cancer Sci 2013;104:266–273. targets for the restoration and reactivation of BRM. 26 Yoshimoto T, Matsubara D, Nakano T, et al. Frequent Oncogene 2014;33:653–664. loss of the expression of multiple subunits of the 41 Gramling S, Rogers C, Liu G, et al. Pharmacologic SWI/SNF complex in large cell carcinoma and reversal of epigenetic silencing of the anticancer pleomorphic carcinoma of the lung. Pathol Int 2015;65: protein BRM: a novel targeted treatment strategy. 595–602. Oncogene 2011;30:3289–3294. 27 Agaimy A. The expanding family of SMARCB1(INI1)- 42 Gramling S, Reisman D. Discovery of BRM targeted deficient neoplasia: implications of phenotype, biolo- therapies: novel reactivation of an anti-cancer gene. gical, and molecular heterogeneity. Adv Anat Pathol Lett Drug Des Discov 2011;8:93–99. 2014;21:394–410. 43 Kahali B, Marquez SB, Thompson KW, et al. 28 Biegel JA, Tan L, Zhang F, et al. Alterations of the Flavonoids from each of the six structural groups hSNF5/INI1 gene in central nervous system atypical reactivate BRM, a possible cofactor for the anticancer teratoid/rhabdoid tumors and renal and extrarenal effects of flavonoids. Carcinogenesis 2014;35: rhabdoid tumors. Clin Cancer Res 2002;8:3461–3467. 2183–2193. 29 Perry A, Fuller CE, Judkins AR, et al. INI1 expression is 44 Jelinic P, Schlappe BA, Conlon N, et al. Concomitant retained in composite rhabdoid tumors, including loss of SMARCA2 and SMARCA4 expression in small rhabdoid meningiomas. Mod Pathol 2005;18:951–958. cell carcinoma of the ovary, hypercalcemic type. Mod 30 Rao Q, Xia Q, Wang Z, et al. Frequent co-inactivation of Pathol 2016;29:60–66. the SWI/SNF subunits SMARCB1, SMARCA2 and 45 Maier S, Wilbertz T, Braun M, et al. SOX2 amplifica- PBRM1 in malignant rhabdoid tumours. Histopathol- tion is a common event in squamous cell carcinomas of ogy 2015;67:121–129. different organ sites. Hum Pathol 2011;42:1078–1088. 31 Jackson EM, Sievert AJ, Gai X, et al. Genomic analysis 46 Karachaliou N, Rosell R, Viteri S. The role of SOX2 in using high-density single nucleotide polymorphism-ba- small cell lung cancer, lung adenocarcinoma and sed oligonucleotide arrays and multiplex ligation-depe- squamous cell carcinoma of the lung. Transl Lung ndent probe amplification provides a comprehensive Cancer Res 2013;2:172–179. analysis of INI1/SMARCB1 in malignant rhabdoid 47 Masai K, Tsuta K, Kawago M, et al. Expression of tumors. Clin Cancer Res 2009;15:1923–1930. squamous cell carcinoma markers and adenocarcinoma 32 Hoot AC, Russo P, Judkins AR, et al. Immunohisto- markers in primary pulmonary neuroendocrine carci- chemical analysis of hSNF5/INI1 distinguishes renal nomas. Appl Immunohistochem Mol Morphol 2013;21: and extra-renal malignant rhabdoid tumors from other 292–297. pediatric soft tissue tumors. Am J Surg Pathol 2004;28: 48 Tatsumori T, Tsuta K, Masai K, et al. p40 is the best 1485–1491. marker for diagnosing pulmonary squamous cell 33 Agaimy A, Rau TT, Hartmann A, et al. SMARCB1 carcinoma: comparison with p63, cytokeratin 5/6, (INI1)-negative rhabdoid carcinomas of the gastroin- desmocollin-3, and sox2. Appl Immunohistochem testinal tract: clinicopathologic and molecular study of Mol Morphol 2014;22:377–382. a highly aggressive variant with literature review. Am J 49 Schaefer I-M, Agaimy A, Fletcher CD, et al. Claudin-4 Surg Pathol 2014;38:910–920. expression distinguishes SWI/SNF complex-deficient 34 Agaimy A, Haller F, Frohnauer J, et al. Pancreatic undifferentiated carcinomas from sarcomas. Mod undifferentiated rhabdoid carcinoma: KRAS alterations Pathol 2017;30:539–548. and SMARCB1 expression status define two subtypes. 50 Fukuoka J, Fujii T, Shih JH, et al. Chromatin remodel- Mod Pathol 2015;28:248–260. ing factors and BRM/BRG1 expression as prognostic 35 Li L, Fan X-S, Xia Q-Y, et al. Concurrent loss of INI1, indicators in non-small cell lung cancer. Clin Cancer PBRM1, and BRM expression in epithelioid sarcoma: Res 2004;10:4314–4324. implications for the cocontributions of multiple 51 Reisman DN, Sciarrotta J, Wang W, et al. Loss of SWI/SNF complex members to pathogenesis. Hum BRG1/BRM in human lung cancer cell lines and Pathol 2014;45:2247–2254. primary lung cancers: correlation with poor prognosis. 36 Hornick JL, Dal Cin P, Fletcher CDM. Loss of INI1 Cancer Res 2003;63:560–566. expression is characteristic of both conventional and 52 Bell EH, Chakraborty AR, Mo X, et al. SMARCA4/BRG1 proximal-type epithelioid sarcoma. Am J Surg Pathol is a novel prognostic biomarker predictive of 2009;33:542–550. cisplatin-based chemotherapy outcomes in resected 37 A phase II, multicenter study of the EZH2 inhibitor non-small cell lung cancer. Clin Cancer Res 2016;22: tazemetostat in adult subjects with INI1-negative tumors 2396–2404. or relapsed/refractory synovial sarcoma - Full Text View 53 Fuller CE, Pfeifer J, Humphrey P, et al. Chromosome - ClinicalTrials.gov. Available from https://clinicaltrials. 22q dosage in composite extrarenal rhabdoid tumors: gov/ct2/show/study/NCT02601950?term = EZH2&rank = clonal evolution or a phenotypic mimic? Hum Pathol 1&show_locs = Y#locn. Accessed March 2 2017. 2001;32:1102–1108. 38 Yamamichi N, Yamamichi-Nishina M, Mizutani T, 54 Samalavicius NE, Stulpinas R, Gasilionis V, et al. et al. The Brm gene suppressed at the post-transc- Rhabdoid carcinoma of the rectum. Ann Coloproctol- riptional level in various human cell lines is inducible ogy 2013;29:252–355. by transient HDAC inhibitor treatment, which 55 Wick MR, Ritter JH, Dehner LP. Malignant rhabdoid exhibits antioncogenic potential. Oncogene 2005;24: tumors: a clinicopathologic review and conceptual 5471–5481. discussion. Semin Diagn Pathol 1995;12:233–248.

Modern Pathology (2017) 30, 1422–1432 SMARCA4/BRG1 loss in thoracic sarcomas 1432 JL Sauter et al

56 Tan A, Mohan GR, Stewart CJR. BRG1-deficient 61 Chan-Penebre E, Armstrong K, Drew A, et al. Selective dedifferentiated endometrioid adenocarcinoma of killing of SMARCA2- and SMARCA4-deficient small the ovary. Pathology 2016;48:82–83. cell carcinoma of the ovary, hypercalcemic type cells 57 Agaimy A, Bertz S, Cheng L, et al. Loss of expression of by inhibition of EZH2: in vitro and in vivo precli- the SWI/SNF complex is a frequent event in undiffer- nical models. Mol Cancer Ther 2017;16:850–860. entiated/dedifferentiated urothelial carcinoma of the 62 Januario T, Ye X, Bainer R, et al. PRC2 mediated urinary tract. Virchows Arch Int J Pathol 2016;469: repression of SMARCA2 predicts for EZH2 inhibitor 321–330. activity in tumors with SWI/SNF mutations. Available 58 Pancione M, Remo A, Sabatino L, et al. Right-sided from www.abstractsonline.com/pp8/#!/4292/presenta rhabdoid colorectal tumors might be related to the tion/1610. serrated pathway. Diagn Pathol 2013;8:31. 63 Rzymski T, Wrobel A, Mikula M, et al. Epigenetic 59 Kim KH, Roberts CWM. Targeting EZH2 in cancer. Nat modulators show differential activity on lung adeno- Med 2016;22:128–134. carcinoma cells with loss-of-function mutations of 60 Margueron R, Reinberg D. The Polycomb complex SWI/SNF protein SMARCA4. Available from www. PRC2 and its mark in life. Nature 2011;469:343–349. abstractsonline.com/pp8/#!/4292/presentation/3803.

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